The objective of this Phase I - Phase II Fast Track Research Proposal is to advance the development of a combined Mass Spectrometric Immunoassay (MSIA) - Bioreactive Mass Spectrometer Probes (BRP) approach to a robust multiplexed technology that will ultimately be used in population screening applications and in studies relevant to cancer research. Individually, the MSIA and BRP technologies had been under development at Intrinsic Bioprobes Inc. for the last several years. Each technology brings a unique perspective to protein analysis. The MSIA approach utilizes affinity captures to selectively retrieve proteins from biological samples for subsequent MALDI-TOF MS analysis. When internal standards are incorporated in the analysis, absolute and/or relative quantitation of the assayed proteins can be achieved. On the other hand, the Bioreactive Probes aid in the thorough structural protein characterization via utilization of MALDI target surface-immobilized enzymes capable of digesting minute amounts of proteins. When combined together, the two technologies can yield a complexed approach that enables sensitive detection, quantitation and structural analysis of specific proteins from complex biological samples. In the Phase I Research, we propose to evaluate and demonstrate the reproducibility and sensitivity of the MSIA technology via execution of well-designed experiments using several model proteins and MSIA assays. In Phase II, we will demonstrate the robustness and the high-throughput capability of the combined MSIA-BRP approach. We will also proceed with the development of additional MSIA-BRP assays for proteins endogenously present in plasma and urine, some of which have been indicated as potential cancer biomarkers. A number of these proteins and assays will be utilized for the development and evaluation of a novel, relative quantitation MSIA approach. And finally, the assays and protocols designed in the Phase I and Phase II will be tested in a large-scale setting (200-300 samples, both urine and plasma) to validate the potential of the MSIA-BRP technology for population screening experiments. The ultimate product resulting from this Phase I-Phase II Research will be a combined MSIA-BRP platform and assays capable of assessing both qualitative and quantitative modulations in proteins analyzed directly from plasma, urine, or other biological fluid or tissue.